The long-term goal of this project is an understanding of the mechanisms that account for CD4+ T cell tolerance to antigens that are presented in the secondary lymphoid organs but not the thymus. Previously, we showed that naive CD4+ T cells that are exposed to model antigens in the secondary lymphoid organs in the absence of inflammation proliferate poorly, then most of the progeny die, and the survivors enter an anergic state characterized by poor lymphokine production. The goal of this application is to establish whether or not a similar series of events accounts for peripheral tolerance to certain natural self-proteins. This has become a pressing issue since the discovery that the AIRE transcription factor drives ectopic expression of extrathymic gene products in the thymus. Thus, it remains possible that the physiological role that peripheral tolerance was thought to play is really played by AIRE-mediated intrathymic tolerance. Here, we will test the hypothesis that peripheral tolerance is physiologically-relevant by using new tools to identify the tolerance mechanisms that apply to two pregnancy-specific proteins, one that appears to be regulated by AIRE and another that does not, and one sperm-specific protein. We will determine whether or not these proteins are immunogenic in mice that have never expressed them in the relevant tissue (e.g., non-pregnant female mice), and become non-immunogenic in mice after expression (e.g., pregnant female mice). The relevant antigenic peptides will be identified and used to produce peptide-MHC II multimers. The multimers will then be used with a sensitive new enrichment method capable of detecting fewer than 100 cells per mouse to enumerate peptide MHC II- specific CD4+ T cells before, during, and after expression of the relevant protein within the polyclonal repertoires of normal mice. This approach should reveal whether or not the relevant CD4+ T cells are deleted, turn into regulatory cells, or become anergic during or after the period when these developmental^ regulated self-proteins are expressed. This approach will then be used in gene-targeted mice to determine whether or not molecules such as Fas and Cbl-b, and others identified by other members of the P01, are involved in the identified tolerance mechanism. Success would provide the first definitive identification of the peripheral tolerance mechanism that applies to a natural self-antigen. This information should help focus future research on the relevant mechanism and shed light on the potential ways that it could fail and lead to autoimmunity.